Density and Distribution of Tetrodotoxin Receptors in Normal and Detubulated Frog Sartorius Muscle ENRIQUEJAIMOVICH, and P A U L H O R O W I C Z

R O Q U E A. V E N O S A , P E T E R S H R A G E R ,

F r o m the D e p a r t m e n t o f Physiology, University of R o c h e s t e r School of M e d i c i n e a n d Dentistry, Rochester, New Y o r k 14642

^ B s T a Ac T Tetrodotoxin (TTX) binding was measured in muscles which were either in normal condition or which had been "detubulated" by glycerol-induced osmotic shock. In both cases the binding of TTX was found to saturate at high TTX concentrations. Maximum binding in normal fibers was 35 pmol/g wet weight, and that figure was reduced to 16 pmol/g after glycerol treatment. The dissociation constant for binding to the surface membrane was 3 nM, which is in the range of values obtained by electrophysiological measurements of the effect of TTX on the maximum rate of rise of the action potential. The results suggest that the dissociation constant in the transverse tubules may be higher than that in the surface. Control experiments indicate that the ef'f'ects of glycerol treatment are limited to the accessibility of the receptors to the toxin and result in no alteration of the affinity of the binding site. TTX receptors in the transverse tubules may be recovered after glycerol treatment by homogenization of the fibers. The measurements suggest that the density of sodium channels in the surface membrane is about 175//zm2 and that in the transverse tubular membrane is 41-52/p.m 2. INTRODUCTION

T h e surface m e m b r a n e o f f r o g skeletal muscle is capable o f c o n d u c t i n g action potentials t h r o u g h the activation o f tetrodotoxin-sensitive sodium channels. It has been suggested that the inward spread o f excitation along the transverse tubules (T system) may be mediated by a similar system (Costantin, 1970; Costantin a n d Taylor, 1971; Gonz~lez-Serratos, 1971; Bezanilla et al., 1972; C a p u t o a n d Dipolo, 1973; Bastian and Nakajima, 1974). T e t r o d o t o x i n ( T T X ) is a highly selective a n d p o t e n t inhibitor o f sodium channels in excitable m e m b r a n e s (Narahashi, 1972). T h e b i n d i n g o f this toxin to cell surfaces has been utilized as a measure o f the density o f s o d i u m channels in a n u m b e r o f p r e p a r a t i o n s (Moore et al., 1967; Keynes et al., 1971; C o l q u h o u n et al., 1972, 1974; Almers a n d Levinson, 1975). Frog sartorius muscle is potentially valuable in these studies because it is c o m p o s e d o f a relatively h o m o g e n e o u s population o f fibers, is susceptible to electrophysiological m e a s u r e m e n t s via intracellular r e c o r d i n g , and allows a disconnection o f the T system f r o m the cell surface. This latter d e c o u p l i n g may be accomplished in Rana pipiens by the use o f glycerol-induced THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 67, 1976 ' p a g e s 399-416

399

400

T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E

67

- 1976

o s m o t i c s h o c k ( H o w e l l a n d J e n d e n , 1967; E i s e n b e r g a n d G a g e , 1967). A f t e r this t r e a t m e n t , f i b e r s r e t a i n t h e ability to c o n d u c t a c t i o n p o t e n t i a l s in t h e s u r f a c e m e m b r a n e , b u t fail to c o n t r a c t . M o r p h o l o g i c a l s t u d i e s h a v e s h o w n t h a t t h e T s y s t e m is n o l o n g e r a c c e s s i b l e to e x t r a c e l l u l a r s o l u t e s ( E i s e n b e r g a n d E i s e n b e r g , 1968; F r a n z i n i - A r m s t r o n g et a l . , 1973). P r o v i d e d t h a t g l y c e r o l t r e a t m e n t d o e s n o t a l t e r t h e t o x i n - r e c e p t o r i n t e r a c t i o n , this p r o c e d u r e m a y b e u s e f u l in d e t e r mining the distribution of sodium channels between surface and transverse t u b u l a r m e m b r a n e s . W e h a v e s t u d i e d t h e b i n d i n g o f T T X to n o r m a l a n d "detubulated" muscles using a bioassay technique. Our results suggest that about o n e - h a l f o f t h e s o d i u m c h a n n e l s in f r o g s a r t o r i u s m u s c l e a r e l o c a t e d in t h e t r a n s v e r s e t u b u l a r s y s t e m . A p r e l i m i n a r y r e p o r t o f this w o r k h a s b e e n p r e s e n t e d ( J a i m o v i c h et al., 1975). METHODS

Freshly isolated sartorius muscles from healthy frogs, R. pipiens, were used t h r o u g h o u t this investigation. Their mean wet weight was 75 -+ 18 mg (1 SD).

Solutions and Reagents The composition o f the standard Ringer's fluid was the following (in mM): NaCI, 115; KCI, 2.5; CaCI2, 1.8; Na2HPO4, 2.15; NaH2PO4, 0.85, p H , 7.2. Low [Na+]0 solutions were made by partial replacement of Na + by choline + in the above solution on a 1:1 basis. In the experiments where low [Na+]o solutions were used D-tubocurarine (10 -s g/ml) was also a d d e d to all solutions. The hypertonic solution (glycerol-Ringer's fluid) used for "detubulation" was p r e p a r e d by adding 400 mM glycerol to standard Ringer's fluid. T h e solution with high [Ca++]o a n d [Mg++]o contained (in mM): NaCI, 115; KCI, 2.5; CaCl2, 5.0; MgCI2, 5.0; Na2HPO4, 1.1; NaH2PO4, 0.4, pH, 7.2. T T X was a d d e d to solutions daily from a refrigerated stock solution of 2 × 10 -5 M. T T X was obtained from Sankyo Co., Tokyo, J a p a n . D-tubocurarine chloride was purchased from Squibb and Sons, Inc., New York. [14C]sucrose and [~4C]inulin were obtained from New England Nuclear, Boston, Mass. All other c o m p o u n d s were reagent grade.

Glycerol Treatment In some muscles the transverse tubular system was disrupted by bathing in glycerolRinger's fluid for 75 min followed by a 75-min exposure to high [Ca++]o and [Mg++]o solution (Howell and J e n d e n , 1967; Eisenberg et al., 1971). Usually at the end of this period no twitch can be recorded, while 90% o f the transverse tubules are disconnected from the surface m e m b r a n e and are inaccessible to extracellular markers (FranziniA r m s t r o n g et al., 1973). After the exposure to high [Ca++]o and [Mg++]0 the muscles were washed for about 1 h in normal Ringer's fluid before measuring T T X binding. This protocol seems to slow the depolarization usually seen in glycerol-treated muscles (Eisenberg et al., 1971; Venosa and Horowicz, 1973).

Electrical and Mechanical Measurements Resting and action potentials were recorded using the method recently described by Stefani and Schmidt (1972) for pyriformis and iliofibularis muscles. This method allows recording in isotonic media without motion artifacts. In the present experiments a sartorius muscle was stretched to 1.2-1.3 times its body length, rolled a r o u n d a Plexiglas rod and placed in a suitable chamber. In this condition, movement associated with a

JAIMOVICH, VENOSA, SHRAGER,AND HOROWlCZ TTX Receptorsin Frog Muscle

401

twitch was considerably r e d u c e d so that single and, in many instances, several action potentials f r o m a single fiber in normal Ringer's fluid could be easily obtained with no apparent damage to either electrode or cell. Conventional glass microelectrodes filled with 3 M KC1 (6-20-MD resistance) were used. They were connected to the input of a cathode follower with negative capacitance compensation. T h e output o f the cathode follower was connected to one channel of a storage oscilloscope. This signal was also electronically differentiated so that both the action potential and its time derivative (V) were displayed on the oscilloscope and photographed. T h e magnitude of the peak twitch tension from muscles in the presence of different solutions was recorded using a different a r r a n g e m e n t which has been described in a previous paper (Venosa, 1974).

Bioassay of Tetrodotoxin T h e bioassay for T T X (Moore et al., 1967; Keynes et al., 1971) was p e r f o r m e d using a method similar to that described by Shrager and Profera (1973), but utilizing a frog sciatic nerve. T h e nerve was desheathed over a 10-15-mm segment and was placed in a Plexiglas chamber designed to isolate a 2-ram portion of the desheathed region between two air gaps. T h e nerve was sealed in place with Vaseline and stimulation and recording o f c o m p o u n d action potentials were by means of external platinum electrodes. One recording electrode was placed at the center of the 2-ram test region. A dose-response curve (Fig. 1) was measured by applying successive 20-pJ aliquots of Ringer's fluid containing known T T X concentrations to the test segment, washing with Ringer's fluid between applications. Equilibrium was usually reached within 15 min and reversibility on washing was consistently close to 100%. 20-/.d aliquots of Ringer's fluid containing unknown concentrations of T T X were applied in similar m a n n e r and the T T X concentration IO0

75

5O

==

~T 25

i

0 0

20

40

60

TTX Concentration (nM)

FIGURE 1. Dose-response curve for T T X bioassay. Percent inhibition of the amplitude of the c o m p o u n d action potential of desheathed frog sciatic nerve is shown as a function of T T X concentration. Each point represents the mean of 4-17 measurements and the vertical bars show the SD.

402

T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y ' V O L U M E 6 7 • 1 9 7 6

determined from the resulting equilibrium height of the c o m p o u n d action potential and the dose response curve (measured for each assay nerve). T h e bioassay p r o c e d u r e was carried out at room temperature.

TTX Binding Procedure From two to four isolated muscles were drained by blotting the tendons with filter paper, and their wet weight d e t e r m i n e d . They were then incubated in 0.2-1.0 ml of Ringer's fluid containing 10-200 nM T T X and with [14C]sucrose (1 mM carrier, 5 x 105 cpm/ml) or [14C]inulin (carrier-free, 5 x 105 cpm/ml) a d d e d as a m a r k e r for the extracellular space. T h e system was kept at room t e m p e r a t u r e (21-23°C) with stirring, and after 30-180 min, samples were taken for the bioassay and radioactive counting. Muscles to be glycerol treated were weighed betore placing them in the glycerol-Ringer's fluid. In some experiments, noted below, muscles were incubated in 100 mi of Ringer's fluid containing T T X , for periods of up to 6 h and then removed, drained as above, and incubated in 0.2-0.3 ml of Ringer's fluid containing [14C]sucrose for 40-60 min. Samples were taken from this solution for bioassay and 14C counting. T h e extracellular fluid volume was d e t e r m i n e d from the dilution of radioactivity after incubation. In some experiments, groups of four muscles were either cut with scissors or were homogenized in several 10-s periods with a cell disrupter ( T e k m a r Co., Cincinnati, Ohio, model SDT 182) in 5-ml Ringer's fluid. In o r d e r to measure the T T X binding, the homogenate was centrifuged for 30 min at 17,000 g and the pellet resuspended by homogenization in 1 ml o f Ringer's fluid containing T T X and [14C]sucrose. After incubation for at least 30 min at room t e m p e r a t u r e , the suspension was centrifuged again and the supernatant assasyed for T T X and 14C. Binding was referred to the original intact wet weight of the muscles. As a control, e.g., for K + leakage, normal and glycerol-treated muscles were incubated in 0.2-1.0 ml o f Ringer's fluid in the absence of T T X for the same period as in the T T X binding experiments. Aliquots from these solutions similar in quantity to those used in binding studies had no effect on the c o m p o u n d action potentials of sciatic nerves p r e p a r e d for assay. RESULTS

Internal Potential Measurements B e f o r e e m b a r k i n g u p o n a s t u d y o f t h e b i n d i n g o f t e t r o d o t o x i n to s a r t o r i u s m u s c l e s , e l e c t r i c a l r e s p o n s e s in t h e p r e s e n c e o f d i f f e r e n t c o n c e n t r a t i o n s o f t h e d r u g w e r e m e a s u r e d . T h e r e s u l t s f r o m t h e s e s t u d i e s p r o v i d e d i n d i c a t i o n s as to t h e c o n c e n t r a t i o n r a n g e to b e s t u d i e d f o r b i n d i n g a n d also p r o v i d e d a p h y s i o l o g ical r e s p o n s e c u r v e to b e c o m p a r e d to t h e b i n d i n g d a t a . Fig. 2 a i l l u s t r a t e s a n a c t i o n p o t e n t i a l a n d its t i m e d e r i v a t i v e in t h e p r e s e n c e o f n o r m a l R i n g e r ' s f l u i d . Fig. 2 b s h o w s s i m i l a r r e c o r d s o b t a i n e d f r o m t h e s a m e m u s c l e a b o u t 20 m i n a f t e r a d d i t i o n o f 12 n M T T X to t h e e x t e r n a l f l u i d . A s t e a d y state was g e n e r a l l y r e a c h e d w i t h i n 15 m i n . T h e o v e r s h o o t a n d t h e m a x i m u m r a t e o f rise o f t h e a c t i o n p o t e n t i a l , Vmax, a r e r e d u c e d a n d t i m e to Vmax is i n c r e a s e d a f t e r e x p o s u r e to T T X . Fig. 2 c a n d d s h o w t h e s a m e s e q u e n c e in g l y c e r o l - t r e a t e d f i b e r s . I n a g r e e m e n t with t h e r e s u l t s o f E i s e n b e r g a n d G a g e (1967) t h e n e g a t i v e a f t e r p o t e n t i a l s e e n in n o r m a l m u s c l e is a b s e n t a f t e r d e t u b u l a t i o n . T h e a c t i o n o f T T X o n t h e s p i k e in g l y c e r o l - t r e a t e d f i b e r s , h o w e v e r , as s e e n in Fig. 2 d, is s i m i l a r to t h a t in n o r m a l m u s c l e .

JAIMOVICH, VENOSA, SHRAGER, AND HOROW1CZ

TTX Receptors in Frog Muscle

403

b

L_

C.

~m5 i

I~m$ i

=I

FIGURE 2. Records of intracellular action potentials (AP) and their time derivatives (I)') from different muscle fibers under the following conditions: (a) normal muscle in the presence of normal Ringer's fluid, (b) same muscle as in a 20 min after addition of 12 nM T T X to the bathing medium, (c) glycerol-treated muscle in normal Ringer's fluid, and (d) same muscle as in c 20 min after adding 12 nM T T X externally. The base line for ~" corresponds to V = 0 for the action potential. Effect of T T X Concentration on the Maximum Rate of Rise of Action Potentials and Twitch Tension In general, the a v e r a g e resting potential o f glycerol-treated fibers was a b o u t 10 m V m o r e positive t h a n that o f n o r m a l fibers (see Venosa a n d Horowicz, 1973). In o r d e r to c o m p a r e electrical p a r a m e t e r s b e f o r e a n d after t r e a t m e n t , in a given muscle, only fibers with internal potentials m o r e negative than - 8 5 m V were analyzed. I n all e x p e r i m e n t s , glycerol-treated muscles failed to twitch u p o n s u p r a m a x i m a l stimulation. F u r t h e r m o r e , it has b e e n shown that after t r e a t m e n t using the p r o c e d u r e s described, 90% o f the transverse tubules are d i s r u p t e d ( F r a n z i n i - A r m s t r o n g et al., 1973). T h e effects of d i f f e r e n t T T X concentrations on l~'max for b o t h n o r m a l a n d glycerol-treated fibers are s u m m a r i z e d in Fig. 3. T h e points r e p r e s e n t m e a n s - 1 SD r e c o r d e d f r o m between 4 a n d 62 fibers for each T T X c o n c e n t r a t i o n . T h e overlap o f the data suggests that t h e r e is no significant change in the effects o f T T X on l;'max a f t e r d e t u b u l a t i o n . T h e T T X c o n c e n t r a t i o n causing a 50% reduction o f f'ma~ is in the r a n g e o f 5-7 nM in both cases. At concentrations o f 20 m M a n d above, no action potentials could be elicited by stimulation. T h e m a g n i t u d e o f the p e a k twitch tension in whole muscles d e c r e a s e d with increasing T T X concentrations in a m a n n e r similar to that o b s e r v e d for f'ma~. T h e twitch is r e d u c e d to o n e - h a l f its n o r m a l value by 8 nM T T X or by r e d u c i n g [Na+]0 to 40% o f its original value. T h e twitch fails either in T T X concentrations above 20 nM or in external m e d i a with less than 25 m M [Na+]o. Tetrodotoxin Binding Since m e a s u r e m e n t s o f b i n d i n g were to be m a d e at equilibrium, we first investigated the d e p e n d e n c e o f T T X b i n d i n g on incubation time, for o u r e x p e r i m e n t a l conditions o f t e m p e r a t u r e a n d stirring. T h e results are shown in Fig. 4, which

404

THE JOURNAL OF GENERAL PHYSIOLOGY'VOLUME 6 7 .

500

1976

,16

4o0 02 I0 4

300

.~

19

200 9 I00

0

'

'

i

0

,5

I0

RIO_

15

20

TTX Concenlrotion (nM)

FIGURE 3. Maximum rate of rise of" the action potential in normal (Q) and glycerol-treated (©) muscles as a function of TTX concentration. The vertical bars represent 1 SD and the numbers next to the points indicate the number of fibers studied. includes data at several different T T X concentrations. In all experiments maximal binding was reached in less than 30 min and this time was t h e r e f o r e taken as the m i n i m u m incubation time. T e t r o d o t o x i n binding was m e a s u r e d in both normal and glycerol-treated muscles. In each e x p e r i m e n t two to four isolated muscles were incubated in a known initial concentration o f T T X for f r o m 30 to 60 rain. T h e equilibrium concentration o f T T X was then measured via the bioassay technique. T o x i n binding (b) was calculated according to Eq. 1. b =

[ T T X ] ~ V - [TTX]rV'

,

(1)

W

where [TTX]i is the known initial concentration o f T T X , [ T T X ] t is the final concentration o f T T X , V is the initial volume o f the incubation m e d i u m containing T T X before exposure to the muscle, V' is obtained by addition o f the extracellular fluid of the muscles m e a s u r e d with [~4C]inulin or [~4C]sucrose (see Methods) to V, and w is the wet weight o f the muscles. Fig. 5 illustrates binding as a function o f the equilibrium T T X concentration. In both normal and detubulated muscles the binding saturates as the toxin concentration is increased. T h e r e seems to be no significant linear, nonsaturable c o m p o n e n t as has been f o u n d in o t h e r preparations using different assay techniques ( C o i q u h o u n et al., 1972, 1974). M a x i m u m T T X binding in normal muscle is on the o r d e r o f 35 pmol/g wet weight, and this is r e d u c e d by about one-half after glycerol treatment. This difference might represent binding to T T X receptors in the transverse tubules.

JAIMOV[CH,

VENOSA,

SHRAGER,

405

TTX Receptors in Frog Muscle

AND H O R O W I C Z

I n o r d e r to test w h e t h e r the reduction in T T X b i n d i n g after glycerol treatm e n t was d u e to an effect o t h e r t h a n d e t u b u l a t i o n , several control e x p e r i m e n t s were carried out. As m e n t i o n e d above, glycerol-treated fibers h a d lower resting potentials t h a n n o r m a l fibers. We tested for the possibility that T T X b i n d i n g may be affected by depolarization or by d a m a g e to the surface m e m b r a n e . Paired muscles were dissected f r o m f o u r frogs. T r a n s v e r s e cuts were m a d e in one m e m b e r o f each pair with scissors so that all fibers were sectioned in at least 10 places. All muscles were soaked in n o r m a l Ringer's fluid for 1 h a n d were then tested for binding. T h e results are given in T a b l e I, row b, a n d show no significant d i f f e r e n c e between u n t r e a t e d a n d cut muscles.

1.0

0.8

o x

~.~

t~0cquO ~

~

~

•

•

o

o

o.6

x

~, 0.4 _° x

02

~ 30

~

, 60 TIME (rain)

~

~ 9O

~

~

l/t---

J20

JSO

Kinetics of T T X binding. Each symbol represents a different experi4. ment done with four muscles in which aliquots were taken at different times from the incubation medium and T T X concentration was measured. The maximum binding for each experiment was used for normalization. The final T T X equilibrium concentrations were, for normal muscle, (©, O) 7 nM, (A) 25 nM, (x) 30 nM; and for glycerol-treated muscle ([7) 30 nM. FIGURE

T o f u r t h e r investigate this m a t t e r , binding e x p e r i m e n t s were carried out with h o m o g e n a t e s (see Methods) f r o m n o r m a l and glycerol-treated muscles. T h e results o f those e x p e r i m e n t s are shown in T a b l e 1, rows c, dl, a n d d2 a n d in Fig. 6. As is seen, this p r o c e d u r e does not m e a s u r a b l y alter the m a x i m u m binding observed in n o r m a l muscles. On the o t h e r h a n d , glycerol-treated muscles exhibited a d r a m a t i c increase in b i n d i n g capacity a f t e r h o m o g e n i z a t i o n with the m a x i m u m b i n d i n g b e i n g a b o u t equal to that f o u n d in n o r m a l muscles. This strongly suggests that the only effect o f glycerol t r e a t m e n t on b i n d i n g is the disconnection of the T system f r o m the external m e d i u m with no a p p a r e n t effect on the T T X receptors. T h e s e e x p e r i m e n t s also suggest that the T T X - r e c e p t o r interaction is indep e n d e n t o f the integrity o f the surface a n d t u b u l a r m e m b r a n e s . F u r t h e r m o r e , it seems that intracellular m e m b r a n e s (sarcoplasmic reticulum, m i t o c h o n d r i a , etc.) are a p p a r e n t l y free o f any significant T T X binding. In a n o t h e r control e x p e r i m e n t we tested for a possible effect o f glycerol on the

406

67 " 1976

T H E J O U R N A L OF G E N E R A L P H ~ ( S I O L O G Y • V O L U M E

40

30

I eD

Q

0 o

Ol~ It)

O0 0

0

I

40

I

0

I

I

80 [20 TTX C0ncef~tration {riM)

160

FIGURE 5. T T X b i n d i n g p e r unit weight as a f u n c t i o n o f T T X c o n c e n t r a t i o n . Each p o i n t was calculated f r o m t h e b i n d i n g to t h r e e to f o u r muscles i n c u b a t e d in 0.2-1.0 ml o f R i n g e r ' s fluid c o n t a i n i n g T T X . • N o r m a l muscles. O Glycerolt r e a t e d muscles. TABLE I

C O N T R O L E X P E R I M E N T S FOR GLYCEROL T R E A T M E N T T T X binding Preparation

Frog muscle

Treatment

Glycerol incubation without osmotic shock

Row reference

T T X concentration

Control

Treated

nM

praol/g

pmol[g

al

41

31

30

a2

21

24

27

30

30

32

35*

37*

Frog muscle

Cutting of all fibers

b

Frog muscle

Homogenization

c

Glycerol-treated frog muscle

Homogenization

dl

57~:

16"

38

d2

65:[:

16"

39

et

80

295

320

e~

7

63

63

Crayfish claw nerves

Glycerol shock

osmotic

* These values correspond to the maximum binding, obtained by linear regression analysis from data at several TTX concentrations (see Fig. 6). $ Concentrations refer to treated muscles only and are in the range where binding to glyceroltreated muscles is maximal.

407

JA1movmH, VENOSA, SHRAOER, AND HOROWICZ TTX Receptors in Frog Muscle

b 40

o®

0 0

30 3

o,

.,-['rT~

o

20

o o

b= 3.L'rr~ r~+[rT~ ( - - )

t-

37 D'T~ b= 13~rT~

0

I

0

J

40

I

t

80

I

J

120

I

1

160

I

0

I

40

l

I

80

t .... )

I

I

120

I

I

160

TTX Conc~n~ (nM)

FIGURE 6. Fit of the binding data to equations shown: (a) glycerol-treated muscles: (©) intact, (~)) homogenate; (b) normal muscles: (O) intact, (Q) homogenate, (ZX) value estimated from the long-term experiment (Table II). The curves are drawn according to the equations. Parameters in a were calculated by linearizing the general form of the binding equation, using the Hanes-Woolf procedure (Hanes, 1932; Endrenyi and Kwong, 1973). Parameters in the equations in b were derived by the procedure described in the text. n u m b e r o f T T X b i n d i n g sites, i n d e p e n d e n t o f detubulation. Several muscles were placed in Ringer's fluid containing 400 m M glycerol in the usual way. T h e r e t u r n to n o r m a l Ringer's fluid was m a d e in f o u r steps, decreasing the concentration o f glycerol by 100 m M in each step. T h e p e a k twitch tension was m o n i t o r e d d u r i n g this p r o c e d u r e a n d was not detectably d i f f e r e n t f r o m that o f u n t r e a t e d muscles. T e t r o d o t o x i n binding to these muscles at two concentrations is given in T a b l e I, rows ai a n d a2, along with data for paired, u n t r e a t e d muscles. Again, no significant d i f f e r e n c e was detected. Finally, we tested for the possible reduction o f b i n d i n g sites in surface m e m b r a n e s in a p r e p a r a t i o n containing no transverse tubules a f t e r glycerol-induced osmotic shock. Claw nerves f r o m 20 crayfish (Procambaru~clarkii) were dissected free by pulling out. H a l f the nerves were placed in n o r m a l Van H a r r e v e l d ' s (1036) saline containing 666 m M glycerol, for 70 min. T h e y were t h e n t r a n s f e r r e d back to n o r m a l Van H a r r e v e l d ' s saline and allowed to stand for 90 min. This t r e a t m e n t r e p r e s e n t s an osmotic shock r o u g h l y equivalent to that applied to f r o g muscle in these studies. After this t r e a t m e n t crayfish nerves r e m a i n excitable, a l t h o u g h the externally rec o r d e d c o m p o u n d action potentials were s o m e w h a t r e d u c e d . T T X b i n d i n g in these nerves was identical to that in u n t r e a t e d , paired controls (Table I, rows ei a n d e2).

408

THE JOURNAL

OF GENERAL

PHYSIOLOGY

" VOLUME

67 • 1976

We have a t t e m p t e d to fit the experimental data to the simplest binding model by assuming that the reaction o f T T X with its receptors is o f the first o r d e r in both surface and tubular m e m b r a n e s , i.e., T T X + Rs-

K,

' Rs'TTX,

(2)

T T X + Rt- K, . R t . T T X ,

(3)

where Rs represents the receptors in surface m e m b r a n e s , Rt represents the receptors in tubular m e m b r a n e s , and Ka and Kt d e n o t e the equilibrium dissociation constants of the surface and tubular m e m b r a n e binding sites, respectively. T h e n , the total binding is given by the equation: b -

Ts[TTX] Ks + [ T T X ]

+

Tt[TTX ] Kt + [ T T X ] '

(4)

where [ T T X ] is the equilibrium concentration o f T T X and T8 and Tt are the maximal binding capacities o f the surface and tubules, respectively. I f we a s s u m e that in g l y c e r o l - t r e a t e d

m u s c l e s the tubular m e m b r a n e s

are

virtually inaccessible, the experimental data can be described by the equation: b,

Ts[TTX] K, + [ T T X ]

(5)

where bs is the binding in the surface m e m b r a n e . Fig. 6 a shows the curve fitted by linear regression analysis o f the data to a r e a r r a n g e d f o r m o f Eq. 5, i.e., [ T T X ] _ K, + 1_ [ T T X ] .

b,,

Ts

(5 a)

Ts

This p r o c e d u r e yields a Ks of 3 +- 2 nM (mean -+ 1 SD) and a surface m e m b r a n e binding capacity, Ts, o f 16 +- 1 pmol/g wet weight o f muscle. From the difference between the binding in normal muscles for T T X concentrations greater than 8 nM (see below) and the values o f bs given by Eq. 5 a Tt o f 19 -+ 1 pmol/g wet weight and Kt o f 12 -+ 5 nM were estimated. In this case the linear regression was less satisfactory (r = 0.97) than that f o u n d for fitting Eq. 5 a to all the data for glycerol-treated muscles (r = 0.99). T h e solid line in Fig. 6 b illustrates the curve obtained by using Eq. 4 and the derived p a r a m e t e r s as described above f r o m the glycerol and normal muscle binding data. We have also a t t e m p t e d to fit the experimental data for normal muscles to a single hyperbolic function of the form: b, =

Tn[TTX] K, + [ T T X ] '

(6)

where the n subscript refers to normal muscle, and all binding sites are taken to be equivalent. Linear regression analysis of all points at toxin concentrations greater than 8 nM yields values o f T, and Kn o f 37 -+ 2 pmol/g wet weight and 13 - 4 nM, respectively. T h e dashed curve in Fig. 6 b is drawn according to Eq. 6, using these parameters. In this case the fit (r = 0.97) was not significantly different f r o m the fit obtained using Eq. 4. H o w e v e r , the equilibrium constant

JAIMOVICH, VENOSA, SHRAGER, AND HOROWICZ

TTX

409

Receptors in Frog M u s c l e

calculated for the single-hyperbola model in n o r m a l muscle (13 +- 4 nM) is significantly different (P < 0.01) f r o m that obtained in glycerol-treated fibers (3 - 2 nM). This result would imply that there is a c h a n g e in the binding characteristics of the T T X r e c e p t o r in the surface after glycerol treatment. However, the electrophysiological data (Fig. 3) suggest no significant alteration o f surface receptor sites after detubulation. In both the double- and single-hyperbola models the derived curves provide fits to the data that are better at high T T X concentrations than at low concentrations. T h e data f r o m h o m o g e n a t e s at these concentrations, however, fall more closely to the curve. O n e possible explanation for the finding that at low T T X concentrations the b i n d i n g in intact muscles is lower than the predicted value, is that the reaction may not have achieved equilibrium at the time of the measurements. In o r d e r to evaluate this possibility an additional e x p e r i m e n t was p e r f o r m e d , designed to test for a very slowly equilibrating c o m p o n e n t o f T T X binding in normal muscle at low toxin concentrations. This e x p e r i m e n t is illustrated by the data in Table II. For the long incubation time r e q u i r e d (6 h), e x p o s u r e to T T X was carried out in a large volume and binding m e a s u r e d after release o f the toxin f r o m the muscle in a small volume o f Ringer's fluid (row I). This proced u r e was followed in o r d e r to avoid possible contamination o f the incubation m e d i u m with K +, etc., and possible deterioration o f the muscle due to anoxia. As a control for the release p r o c e d u r e an e x p e r i m e n t was carried out with an initial incubation for a short time, at a high T T X concentration, followed by a release phase similar to that used above (row II). Four n o r m a l muscles were incubated at r o o m t e m p e r a t u r e for 6 h in a large volume (100 ml) o f Ringer's fluid containing 10 nM T T X (t/ptake phase). T h e muscles were then drained a n d t r a n s f e r r e d to a small volume (0.2 ml) o f T T X - f r e e Ringer's fluid containing [~4C]sucrose, and incubated for 60 min with stirring (release phase). At the end o f the release phase a new equilibrium was established between b o u n d and u n b o u n d T T X . Aliquots o f 20-/~1 bathing fluid were then assayed. Controls for this long-term, large v o l u m e uptake and subsequent release p r o c e d u r e consisted o f incubation TABLE

II

LONG-TERM BINDING EXPERIMENT ON NORMAL MUSCLE

Row reference

Incubation time

Equilibrium TTX concentration, uptake phase

~n

nM

I*

360

10

30

40

TTX bound pmol ~ -

30

Equilibrium TTX concentration, release phase

TTX released

nM

pmol~

7

10.5

7.5

21

* Muscles were incubated in 100 ml of Ringer's fluid containing 10 nM TTX for 6 h, then transferred to 0.2 ml of TTX-free Ringer's fluid for 60 min. Aliquots of the final incubation medium were assayed. Muscles were incubated in 0.3 ml of Ringer's fluid containing 50 nM TTX for 30 min and aliquots were removed and assayed. The muscles were then transferred to 0.3 ml of TTX-free Ringer's fluid for 60 min and aliquots again removed for assay.

410

THE

JOURNAL

OF

GENERAL

PHYSIOLOGY

• VOLUME

6 7 . 1976

o f n o r m a l muscles at high T T X concentrations for s h o r t e r times, in small volumes. Binding at this stage was m e a s u r e d using an aliquot o f the incubation m e d i u m . T h e muscles were t h e n t r a n s f e r r e d to a small volume o f T T X - f r e e Ringer's fluid containing [14C]sucrose, a n d at the e n d o f the release phase, aliquots o f the b a t h i n g m e d i u m were assayed for T T X . T h e s e control experim e n t s were designed to result in a final T T X equilibrium c o n c e n t r a t i o n a f t e r release (ca. 7 nM) similar to that obtained in the l o n g - t e r m e x p e r i m e n t with the lower concentration o f T T X . T h e binding e x p e c t e d after a s h o r t - t e r m incubation with an equilibrium concentration o f 7 nM T T X is 9 - 3 p m o l / g (Fig. 5). This value o f b i n d i n g agrees well with that f o u n d by taking the d i f f e r e n c e between the a m o u n t o f T T X b o u n d a f t e r s h o r t - t e r m incubation (30 pmol/g; T a b l e I I , row 2) and that released when the muscles were subsequently reincubated in a T T X - f r e e m e d i u m (21 pmol/g). This suggests that binding a f t e r 6 h in 10 nM T T X was on the o r d e r o f 20 p m o l / g (10.5 + 9 pmol/g; T a b l e I I , row I), which is not far f r o m the value o f 20.5 p m o l / g p r e d i c t e d by the equation given in Fig. 6 b for a T T X concentration o f 10 nM, f r o m the s h o r t - t e r m e x p e r i m e n t s . It should be noted, h o w e v e r , that the curve which has b e e n fit to the data f r o m intact muscles for s h o r t - t e r m incubations at high T T X concentrations predicts a higher T T X binding than that actually m e a s u r e d at 10w concentrations o f T T X . This implies that the binding m e a s u r e d at low T T X concentrations has not r e a c h e d equilibrium values. This is also suggested by the fact that the b i n d i n g obtained with h o m o g e n i z e d muscles at low T T X concentrations was h i g h e r than that o f intact muscles a n d comes closer to the curve (Fig. 6 b). On the o t h e r h a n d , the kinetic e x p e r i m e n t s illustrated in Fig. 4 seem to indicate that equilibrium is a p p a r e n t l y r e a c h e d within 30 min at both high a n d low concentrations. T h e p r o b a b l e e x p l a n a t i o n for this discrepancy is that the scatter in the m e a s u r e m e n t s precludes an accurate m e a s u r e m e n t o f a small, slow c o m p o n e n t in the b i n d i n g at low T T X concentrations using intact muscles. D I S C U S S I O N

Effect of Glycerol Treatment on TTX Binding T h e results o f these e x p e r i m e n t s indicate that 55% of the T T X b i n d i n g sites in f r o g sartorius muscle is eliminated after disconnection o f the T system by glycerol osmotic shock. T h e glycerol t r e a t m e n t e m p l o y e d h e r e is known to u n c o u p l e contraction f r o m excitation by i n t e r r u p t i n g the continuity between surface and transverse t u b u l a r m e m b r a n e s (Howell a n d J e n d e n , 1967; Gage and Eisenberg, 1969). It has been shown that in glycerol-treated muscles the T tubules which r e m a i n accessible to l a n t h a n u m (an extracellular m a r k e r ) r e p r e sent 10% o f the a m o u n t f o u n d in n o r m a l fibers ( F r a n z i n i - A r m s t r o n g et al., 1973). We have c o n s i d e r e d the possibility that s o m e o f the reduction in binding after glycerol t r e a t m e n t m i g h t be due to effects o t h e r than detubulation. Muscles e x p o s e d to identical glycerol concentrations, but r e t u r n e d to n o r m a l Ringer's fluid in a m a n n e r designed to avoid strong osmotic shock retained the ability to contract a n d also retained full T T X b i n d i n g capacity. H o m o g e n i z a t i o n of d e t u b u l a t e d fibers allows the recovery of the fraction o f T T X b i n d i n g lost

JAIMOVmH, VENOSA,Srl~,GZ~, AND HoRowmz TTX Receptors in Frog Muscle

411

after glycerol t r e a t m e n t , suggesting that this latter p r o c e d u r e p r e s e r v e s the integrity o f the T T X r e c e p t o r s , while r e n d e r i n g s o m e o f t h e m inaccessible to the toxin. T h i s conclusion is f u r t h e r s u p p o r t e d by the control e x p e r i m e n t s on n o n m y e l i n a t e d nerve, which resulted in no loss o f T T X b i n d i n g after an equivalent glycerol-induced osmotic shock. Also, the dissociation constant after d e t u b u l a u o n is close to the electrophysiologically derived value, a n d is o f the s a m e o r d e r as that f o u n d in n e r v e fibers ( C u e r v o a n d A d e l m a n , 1970; Hille, 1968; Schwarz et al., 1973). Glycerol t r e a t m e n t is k n o w n to result in partial depolarization o f s o m e fibers. H o w e v e r , depolarization itself, p r o d u c e d either by high external p o t a s s i u m concentrations (Almers a n d Levinson, 1975) or by cutting or by h o m o g e n i z a t i o n failed to r e d u c e toxin binding. T h e results o f these control e x p e r i m e n t s t h e r e f o r e suggest that the d i f f e r e n c e in T T X b i n d i n g between n o r m a l a n d glycerol-treated muscle is a m e a s u r e of the b i n d i n g capacity o f the t r a n s v e r s e t u b u l a r m e m b r a n e and that the T T X b i n d i n g m e a s u r e d in d e t u b u l a t e d muscles mainly r e p r e s e n t s b i n d i n g to the surface m e m b r a n e . Further, these data suggest that the physical integrity o f the surface a n d T-system m e m b r a n e s is not essential for toxin binding, a n d that o t h e r cellular c o m p o nents, such as the sarcoplasmic reticulum a n d m i t o c h o n d r i a , lack r e c e p t o r s for TTX.

Specificity and Stoichiometry of T T X Binding DETUBULATED FIBERS T h e b i n d i n g o f T T X to glycerol-treated muscle saturates at a T T X c o n c e n t r a t i o n within one o r d e r o f m a g n i t u d e o f that r e q u i r e d for 50% r e d u c t i o n o f l/max. T h e s e data can be fitted by a single hyperbolic function with a dissociation constant close to that p r e d i c t e d by electrophysiological measu r e m e n t s . T h i s result agrees well with the data o b t a i n e d in n e r v e fibers (Hille, 1968; C u e r v o a n d A d e l m a n , 1970; Schwarz et al., 1973) that suggest a 1:1 interaction between T T X a n d its r e c e p t o r . Within the r a n g e o f toxin concentrations used we find no indication o f a linear, n o n s a t u r a b l e c o m p o n e n t o f binding, such as that described in o t h e r p r e p a r a t i o n s , using [ 3 H ] T T X ( C o l q u h o u n et al., 1972, 1974). For a discussion o f this linear c o m p o n e n t see Almers a n d Levinson (1975), Levinson (1975), a n d C o l q u h o u n et al. (1975). NORMAL FIBERS In n o r m a l muscles T T X b i n d i n g is also f o u n d to saturate at concentrations within o n e o r d e r o f m a g n i t u d e of the physiologically active range. T h e data could be fit a b o u t equally well by either a single- or doubleh y p e r b o l a model. H o w e v e r the f o r m e r case, in which tubule a n d surface receptor sites are t a k e n to be identical yields a value for the equilibrium constant in n o r m a l muscle significantly d i f f e r e n t f r o m that in d e t u b u l a t e d fibers. Since the electrophysiological data suggest that surface r e c e p t o r s are u n a l t e r e d by glycerol t r e a t m e n t , we consider the b i n d i n g m o d e l with d i f f e r e n t a p p a r e n t equilibrium constants for surface a n d tubule sites to be m o r e probable. O w i n g to the scatter in the data at low T T X concentrations a n d the uncertainty o f having achieved equilibrium at these concentrations, it is not possible to reach a definite conclusion c o n c e r i n g the stoichiometry o f T T X b i n d i n g in the T system. T h e simplest a s s u m p t i o n is to consider the stoichiometry to be the same as that for the surface m e m b r a n e , i.e., 1:1. H o w e v e r , we are t h e n led to the conclusion that the

412

THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 6 7 "

1976

a p p a r e n t dissociation constant in the transverse tubules is different f r o m that in the surface. This does not necessarily imply that the receptor for T T X is a different chemical entity in the two cases. T h e local e n v i r o n m e n t s u r r o u n d i n g the receptor may vary due to different densities of fixed surface charge, the presence o f adjacent m e m b r a n e s (e.g., terminal cisternae), or variation in surface forces due to differing geometry.

Density and Distribution of TTX Receptors We measure a m a x i m u m T T X binding in n o r m a l muscle o f 35 p m o l / g wet weight (Table I I I ) . This value may be c o m p a r e d with that recently r e p o r t e d by Almers and Levinson (1975) o f 22 pmol/g. T h e difference in these results may be accounted for by the different surface-to-volume ratios o f the muscle fibers TABLE

Ill

DENSITY AND AFFINITY OF TTX RECEPTORS IN SURFACE AND TRANSVERSE TUBULAR MEMBRANES

Normal muscle Glycerol-treated muscle Normal minus glyceroltreated muscle

TTX binding

[TTX] for half saturation

Density of binding sites

pmol/g

nM

molecules/~ z

35±2 16± 1 19± 1

3±2 12±5

175± 11" 41-52:[:

All values are obtained from equations used to fit the data of Fig. 6. * Surface membrane area = 552 cm~/gof drained muscle (Venosa, 1974). :~The range in density values reflects the possible range of tubular membrane area for fibers with an average diameter equal to 55 t~m (2,200-2,800 cm2/g of drained muscle) (Peachey, 1965; Hodgkin and Nakajima, 1972). employed. T h e average fiber diameter in the sartorii used in these experiments was approximately 55/zm, while that in the work o f Almers a n d Levinson (1975) was 80/~m. I f binding is r e f e r r e d to surface area, assuming cylindrical g e o m e t r y (and neglecting the T system) rather than wet weight, the data for normal muscle are identical in the two cases (ca. 380 sites/tzm2). C o l q u h o u n et al. (1974) have r e p o r t e d [3H]TTX binding to rat d i a p h r a g m muscle with an estimated specific c o m p o n e n t o f 2.5 pmol/g. T h e average fiber diameter in this p r e p a r a tion was o f the o r d e r o f 40 tzm. Aside f r o m the species difference, we have no explanation for the r a t h e r large difference in the a p p a r e n t receptor density in rat d i a p h r a g m as c o m p a r e d with that in frog sartorius muscle. T h e r e have been a n u m b e r o f estimates o f the density o f T T X receptors in nerve m e m b r a n e s . O f particular interest here are those m a d e on relatively hom o g e n e o u s preparations. In the very small fibers o f garfish olfactory nerve (diameter, 0.25 tzm) values of 3.9 sites/~m 2 (Benzer and Raftery, 1972) to 5.6 sites/ ~ m 2 ( C o l q u h o u n et al., 1972, 1974) have been r e p o r t e d . I n squid giant axons (diameter 400-600 t~m) T T X binding has been measured at 553 sites/t~m 2 (Levinson and Meres, 1975). O u r measurements suggest a value o f 175 sites/g~m 2 (Table III) for the surface m e m b r a n e o f frog muscle (diameter 55 lzm). This estimate,

JAIMOVICH, VENOSA,SHRAGER,AND HOROWlCZ TTX Receptors in Frog Muscle

413

u n c o r r e c t e d for the small fraction o f transverse tubules r e m a i n i n g a f t e r glycerol t r e a t m e n t , thus falls b e t w e e n values for smaller a n d larger n e r v e fibers. T h e r e have also b e e n m a n y estimates o f dissociation constants in n e r v e b o t h f r o m T T X b i n d i n g a n d f r o m electrophysiological studies, a n d almost all fall in the r a n g e 310 nM (Hille, 1968; C u e r v o a n d A d e l m a n , 1970; C o l q u h o u n a n d Ritchie, 1972; Schwarz et al., 1973). O u r m e a s u r e d value o f 3 nM for the surface m e m b r a n e thus agrees well with those studies, Values o f 41-52 sites//~m 2 can be calculated for the b i n d i n g density in tubular m e m b r a n e s f r o m the d i f f e r e n c e in T T X b i n d i n g between n o r m a l a n d glyceroltreated fibers, a n d f r o m the a r e a o f t u b u l a r m e m b r a n e for the a v e r a g e fiber d i a m e t e r (Table I I I ) . I f one assumes that each T T X b i n d i n g site is associated with one s o d i u m channel a n d that the electrical characteristics o f the channels are the s a m e for t u b u l a r a n d surface locations, t h e n the density o f s o d i u m channels a n d the limiting s o d i u m c o n d u c t a n c e in the tubular walls is a b o u t oneq u a r t e r that in the fiber surface. F r o m the n u m e r i c a l construction o f action potentials in f r o g sartorius muscle Adrian a n d Peachey (1973) c o n c l u d e d that the ratio o f s o d i u m channel density in the tubules to that in the surface is 1:20. A l t h o u g h this ratio is smaller by a factor o f 5 t h a n that based on the T T X binding figures in this r e p o r t , n u m e r i c a l r e c o n s t r u c t i o n o f action potentials permits a wide choice o f p a r a m e t e r s for any given m o d e l network. In addition, the m o d e l n e t w o r k chosen for n u m e r i c a l analysis influences the choice of a p a r a m e t e r such as the limiting s o d i u m c o n d u c t a n c e in the transverse tubules. It is likely, t h e r e f o r e , that a ratio o f 1:4 for the density o f s o d i u m channels in the tubules to that in the surface can be a c c o m m o d a t e d in theoretical a n d n u m e r i c a l r e c o n s t r u c t i o n o f action potentials. T h e time-course o f T T X b i n d i n g in o u r e x p e r i m e n t s was significantly faster than that r e p o r t e d by A l m e r s a n d Levinson (1975). F r o m their Fig. 3 the u p t a k e o f the toxin by muscles e x p o s e d to 10 nM T T X at 4°C has a half time o f a b o u t 17 min. O u r e x p e r i m e n t s , on the o t h e r h a n d , carried out at 21-23°C, have a m e a n half time o f a p p r o x i m a t e l y 5 min (Fig. 4), which is close to the half time for the e x c h a n g e o f Tris + for Na + (2.2 min) by diffusion in the same p r e p a r a t i o n , a n d in the same t e m p e r a t u r e r a n g e (Venosa, 1974). In two control e x p e r i m e n t s , with the incubation step carried out at 2°C, the rate o f b i n d i n g was considerably slowed, with an estimated h a l f time o f 15 rain, in reasonable a g r e e m e n t with the results o f Almers a n d Levinson (1975). Since c h a n g e s in t e m p e r a t u r e should have only a small effect on the diffusion constant o f the d r u g this suggests that the rate o f reaction between the toxin a n d its b i n d i n g site may be a significant factor in d e t e r m i n i n g the kinetics of binding at low t e m p e r a t u r e s . O n e final point worth c o n s i d e r i n g is the m a x i m u m c o n d u c t a n c e o f a single s o d i u m channel. Almers a n d Levinson (1975) using all the m e a s u r e d T T X binding sites, without allowance f o r sites in the T system, a n d the m a x i m u m s o d i u m c o n d u c t a n c e m e a s u r e m e n t s by A d r i a n et al. (1970) a n d I l d e f o n s e a n d Roy (1972) calculate the limiting s o d i u m c o n d u c t a n c e to be 0.6-1.4 p m h o . T h e s e values will be larger if t u b u l a r sites are to a d e g r e e electrically isolated a n d t h e r e f o r e do not c o n t r i b u t e their full share to the p e a k s o d i u m c o n d u c t a n c e o f muscle m e m b r a n e . In the limit where t u b u l a r sites are completely isolated o u r

414

THE JOURNAL OF GENERALPHYSIOLOGY'VOLUME67"1976

results s u g g e s t t h a t t h e s e v a l u e s s o u l d be m u l t i p l i e d by a f a c t o r o f a b o u t 2. T h i s b r i n g s t h e c a l c u l a t e d s i n g l e - c h a n n e l c o n d u c t a n c e i n m u s c l e closer to t h e m o r e r e c e n t e s t i m a t e s for this p a r a m e t e r in n e r v e ( K e y n e s a n d Rojas, 1974; L e v i n s o n a n d Meves, 1975). This work has been supported by grants 5-P01-NS10981 and 5-R01-NS10500 from the National Institutes of Health.

Receivedfor publication 8 August 1975. REFERENCES

ADRIAN, R. H., W. K. CHANDLER,and A. L. HODGKIN. 1970. Voltage clamp experiments in striated muscle fibres.J. Physiol. (Lond.). 208:607-644. ADRIAN, R. H., and L. D. PEACHEY. 1973. Reconstruction of the action potential of frog sartorius muscle.J. Physiol. (Lond.). 235:103-131. ALMERS, W., and S. R. LEVINSON. 1975. Tetrodotoxin b i n d i n g to normal and depolarized frog muscle and the conductance of a single sodium channel. J. Physiol. (Lond.). 247:483-509. BASTIAN,J., and S. NAKAJIMA. 1974. Action potential in the transverse tubules and its role in the activation of skeletal muscle. J. Gen. Physiol. 65:257-278. BENZER, T. I., and M. A. RAFTERV. 1972. Partial characterization of a tetrodotoxin binding component from nerve m e m b r a n e . Proc. Natl. Acad. Sci. U.S.A. 69:3634-3637. BEZANILLA, F., C. CAPUTO, H. GONZALEZ-SERRATOS,and R. A. VENOSA. 1972. Sodium dependence of the inward spread of activation in isolated twitch muscle fibres of the frog.J. Physiol. (Lond.). 223:507-523. CAPUTO, C., and R. DIPoLo. 1973. Ionic diffusion delays in the transverse tubules of frog twitch muscle fibres. J. Physiol. (Lond.). 229:547-557. COLQUHOUN, D., R. HENDrRSON, and J. M. RITCHIE. 1972. T h e binding of labelled tetrodotoxin to non-myelinated nerve fibres. J. Physiol. (Lond.). 227:95-126. COLQUHOUN, D., R. HENDERSON, and J. M. RITCHIE. 1975. The purity of tritiated tetrodotoxin as determined by bioassay. Discussion of S. R. Levinson. Philos. Trans. R. Soc. Lond. B Biol. Sci. 270:337-348. COLQUHOUN, D., H. P. RANG, and J. M. RITCHIE. 1974. T h e binding of tetrodotoxin and ot-bungarotoxin to normal and denervated mammalian muscle. J. Physiol. (Lond.). 240:199-226. COLQUHOUN, D., and J. M. RtTCHIE. 1972. The kinetics of the interaction between tetrodotoxin and mammalian non-myelinated nerve fibers. Mol. Pharmacol. 8:285292. COSTANTIN, L. L. 1970. T h e role of sodium c u r r e n t in the radial spread of contraction in frog muscle fibers. J. Gen. Physiol. 55:703-715. COSTANTIN, L. L., and S. R. TAYLOR. 1971. Active and passive shortening in voltageclamped frog muscle fibres.J. Physiol. (Lond.). 218:13-15P. CUrRVO, L. A., and W. J. ADELMAN,JR. 1970. Equilibrium and kinetic properties of the interaction between tetrodotoxin and the excitable m e m b r a n e of the squid giant axon. J. Gen. Physiol. 55:309-335. EISENBERG, B., and R. S. E1SENBERG. 1968. Selective disruption of the sarcotubular system in frog sartorius muscle. J. Cell. Biol. 39:451-467. EISENBERG, R. S., and P. W. GAGE. 1967. Frog skeletal muscle fibers: Changes in electrical

JAIMOVICH, VENOSA,SHRAGER, AND HOnOWlCZ TTX Receptors in Frog Muscle

415

properties after disruption of transverse tubular system. Science (Wash. D.C.). 158:17001701. EISENBERG, R. S., J. N. HOWELL, and P. C. VAUGHAN. 1971. T h e maintenance of resting potentials in glycerol-treated muscle fibres. J. Physiol. (Lond.). 215:95-102. ENDRENYI, L., and F. H. F. KWONG. 1973. Design and analysis of hyperbolic kinetic and binding experiments. In Analysis and Simulation o f Biochemical Systems. H. C. H e m p e r and B. Hess, editors. North Holland, A m s t e r d a m . 219-237. FRANZINI-ARMSTRONG, C., R. A. VENOSA, a n d P. HoRowlcz. 1973. Morphology and accessibility o f the transverse tubular system in frog sartorius muscle after glycerol t r e a t m e n t . J . Membr. Biol. 14:197-212. GAGE, P. W., and R. S. EISENBERG. 1969. Capacitance of the surface and transverse tubular membrane of frog sartorius muscle fibers.J. Gen. Physiol. 53:265-278. GONZALEZ-SERRATOS, H. 1971. Inward spread of activation in vertebrate muscle fibers.J. Physiol. (Lond.). 212:777-799. HANES, C. S. 1932. T h e effect of starch concentration upon the velocity of hydrolysis by the amylase o f g e r m i n a t e d barley. Biochem. J. 26:1406-1421. HILLE, B. 1968. Pharmacological modifications of the sodium channels o f frog nerve. J. Gen. Physiol. 51:199-219. HODGKIN, A. I., and S. NAKAJIMA. 1972. Analysis of membrane capacity in frog muscle.J. Physiol. (Lond.). 221:121-136. HOWELL, J. N., and D. J. JENOEN. 1967. T-tubules of skeletal muscle: Morphological alterations which interrupt excitation contraction coupling. Fed. Proc. 26:553. (Abstr.). ILDEFONSE, M., and G. Roy. 1972. Kinetic properties o f the sodium current in striated muscle fibres on the basis of the Hodgkin-Huxley theory.J. Physiol. (Lond.). 227:419-431. JAIMOWCH, E., R. A. VENOSA, P. SHRAGF.R,and P. HoRowIcz. 1975. Tetrodotoxin (TTX) binding in normal and "detubulated" frog sartorius muscle. Biophys. J. 15:255a. (Abstr.). KEYNES, R. D., J. M. RITCmE, and E. RoJAs. 1971. T h e binding of tetrodotoxin to nerve membranes. J. Physiol. (Lond.). 213:235-254. KEYNES, R. D., and E. RojAs. 1974. Kinetics and steady-state properties of the charged system controlling sodium conductance in the squid giant axon. J. Physiol. (Lond.). 239:393-434. LEVINSON, S. R. 1975. T h e purity of tritiated tetrodotoxin as determined by bioassay. Philos. Trans. R. Soc. Lond. B Biol. Sci. 270:337-348. LEVINSON, S. R., and H. MEvEs. 1975. T h e binding of tritiated tetrodotoxin to squid giant axons. Philos. Trans. R. Soc. Lond. B Biol. Sci. 270:349-352. MOORE, J. W., T. NARAHASHI, and T. I. SHAW. 1967. An u p p e r limit to the number o f sodium channels in nerve m e m b r a n e ? J . Physiol. (Lond.). 188:99-105. NARAHASHI, T. 1972. Mechanism of action of tetrodotoxin and saxitoxin on excitable membranes. Fed. Proc. 31:1124-1132. PEACHEV, L. D. 1965. T h e sarcoplasmic reticulum and transverse tubules o f the frog's sartorius. J. Cell Biol. 25:209-232. SrmAGER, P., and C. PROFEnA. 1973. Inhibition o f the r e c e p t o r for tetrodotoxin in nerve membranes by reagents modifying carboxyl groups. Biochim. Biophys. Acta. 318:141-146. SCrZWARZ, J. R., W. ULBRICHT, and H. H. WAGNER. 1973. T h e rate o f action o f tetrodotoxin on myelinated nerve fibres of Xenopus laevis and Rana esculenta. J. Physiol. (Lond.). 233:167-194. STEFANI, E., and H. SCH~IIDT. 1972. A convenient m e t h o d for r e p e a t e d intracellular

416

THE JOURNAL OF GENERALPHYSIOLOGY'VOLUME67" 1976

recording of action potentials from the same muscle fibre without m e m b r a n e damage.

Pfluegers Arch. Eur. J. Physiol. 354:276-278. VAN HARREVELD, A. 1936. Physiological saline for crayfish. Proc. Soc. Exp. Biol. Med. $'1:428-432. VENOSA, R. A. 1974. Inward movement of sodium ions in resting and stimulated frog's sartorius muscle.J. Physiol. (Lond.). 241:155-173. VENOSA, R. A., and P. HOROWICZ. 1973. Effects on sodium efflux of treating frog sartorius muscle with hypertonic glycerol solutions. J. Membr. Biol. 14:33-56.